Orbit/MAST is present during early embryogenesis, but it is highly expressed during late embryogenesis, in larval brains and ovaries, and significantly reduced in testes (Lemos, 2000).
To determine the intracellular localization of Mast during different stages of the cell cycle, anti Mast antibody was used for indirect immunofluorescence in S2 Drosophila tissue culture cells. At interphase, Mast is focused on MTOCs and shows a punctuate pattern co-localizing with alpha-tubulin. At prophase, Mast accumulates at the MTOCs, as shown by alpha-tubulin co-staining. Double immunostaining with either anti-centrosomin (CNN) or gamma-tubulin antibodies also shows co-localization with Mast. In some cells, Mast also associates with a rod-like structure present in interphase or mitotic cells. This structure is stained by antibody when cells are prepared by different fixation methods and in different cell types. At metaphase, Mast localizes to centrosomes, the mitotic spindle and centromeres, maintaining this localization during anaphase A. Later, at anaphase B, Mast appears concentrated at the spindle midzone, associated with polar MTs, and shows faint centrosomal localization until late telophase. During very late telophase, it localizes at either side of the midbody, and centrosomal localization is barely detectable. After cytokinesis, Mast can be seen associated again with the rod-like structure. This structure might correspond to the remains of the midbody (Lemos, 2000).
An EGFP-Mast fusion protein follows a pattern of localization in both cell types similar to that described for the immunolocalization of Mast. In interphase, EGFP-Mast signal is strongly associated with MTOCs and with a fibrillar network that resembles MT bundles. This extensive fibrillar network is not observed in transfected cells treated with colchicine. At prophase, the protein localizes to the cytoplasm and shows accumulation at the centrosome. Later, during prometaphase/metaphase, spindle association is clearly evident, as well as localization to the centrosomes and centromeres. During early anaphase, EGFP-Mast appears more diffuse, although, at later stages, centrosomes, spindle MTs and the spindle midzone show accumulation of the protein. In telophase, both S2 and HeLa cells show EGFP signal at the midbody and centrosomes. These results indicate that Mast associates with MTs and centrosomes during most of the cell cycle but undergoes accumulation in additional structures during mitosis (Lemos, 2000).
If Orbit/MAST plays a direct role in axonal navigation, the protein would be expected to localize within growth cones. Previous characterization of Orbit/MAST expression focused on cell division; however, no description of Orbit/MAST expression in differentiating neurons exists. Using in situ hybridization, it was asked if orbit/MAST is transcribed in the CNS during the late embryonic stages when axon pathways are constructed. At early stages of development, orbit/MAST RNA expression is quite broad. However, during the key stages of neuronal differentiation (stages 12 through 17), the signal accumulates in the developing CNS (Lee, 2004).
To investigate localization of endogenous protein, anti-Orbit/MAST antibodies were used to stain the embryonic CNS; Orbit/MAST immunoreactivity accumulates largely within the neuropil, consistent with localization within axons and growth cones. To obtain higher-resolution images of growth cones that require Orbit/MAST, motor growth cones were examined, visualized with the membrane marker FasII. Orbit/MAST localizes along motor axon shafts as they extend through the periphery. Although Orbit/MAST was also expressed in embryonic muscles, three-dimensional reconstruction of confocal images after deconvolution shows that a substantial amount of Orbit/MAST is contained within the growth cone perimeter as defined by Fas II. Interestingly, mesodermal Orbit/MAST puncta seemed to be most intense at sites of motor nerve contact (Lee, 2004).
To confirm that Orbit/MAST accumulates in growth cones, an Orbit/MAST-GFP fusion protein was expressed in neurons. This construct fully rescues the orbit/MAST mutant defects. Explants of the transgenic CNS were made and axons were grown alone on a glass substrate. In this assay, Orbit/MAST localized along the axon and accumulated at the expanded tips of processes, correlating with the distal ends of axonal microtubules. Taken together, these findings demonstrate that Orbit/MAST protein is in the right location to play a direct role in growth cone turning. However, due to the small size of Drosophila growth cones, high-resolution images of Orbit/MAST subcellular localization and dynamics are difficult to obtain in this system. Thus, larger vertebrate growth cones were used for the next level of analysis (Lee, 2004).
The vertebrate Orbit/MAST ortholog CLASP is known to localize along microtubules and at their plus ends in nonneuronal cells (Akhmanova, 2001); however, its behavior in vertebrate growth cones is unknown. GFP-CLASP fusion proteins that were expressed in developing Xenopus spinal cord neurons were used to examine CLASP localization and dynamics. GFP-CLASP accumulates in comet-shaped dashes that localize to the tips of microtubules labeled with Rhodamine-conjugated Tubulin subunits in motile growth cones. Lower levels of GFP-CLASP were also seen along the length of microtubules and diffusely in the cytoplasm. When the numbers of CLASP-positive dashes were compared in growth cones and axon shafts, it was evident that CLASP was preferentially localized to the growth cone, consistent with a function specialized for axon growth or navigation. Another plus end tracking protein, CLIP-170, showed far less preference for the growth cone and highlighted the presence of microtubule plus end growth along the axon shaft , as reported for CLIP-170 and EB-3 in Purkinje cells. These findings suggest that CLASP identifies a subset of microtubules within the growth cone (Lee, 2004).
The CLASP and CLIP-170 dashes both exhibit movement predominantly toward the growth cone leading edge. Consistent with growing plus end tracking behavior, GFP-CLASP dashes moved at an average of 9.7 ± 2.1 microm/min, matching previously recorded microtubule growth rates in Xenopus neurons. Microtubules that underwent catastrophe and retracted lost their GFP-CLASP signal. Thus, the Orbit/MAST/CLASP protein associates preferentially with dynamic microtubule domains (Lee, 2004).
Further imaging of GFP-CLASP dynamics has revealed that CLASP identifies a subset of microtubules that probe and penetrate the growth cone leading edge where guidance signals influence actin assembly. Tracking of individual GFP-CLASP dashes showed that some of the microtubule plus ends that accumulate CLASP extend into individual filopodia (overlay of many time points illustrates the progressive movement into several filopodia). Often, several microtubule plus ends were observed to follow the same path, as if guided by actin structures in the periphery. Interestingly, GFP-CLASP-positive microtubules could been seen to bend and pause and then conform to a novel path as they entered the growth cone periphery, as was previously described for pioneer microtubules interacting with actin filaments in the growth cone. Indeed, when actin and GFP-CLASP were imaged simultaneously, microtubules could be seen to track along actin bundles emanating from individual filopodia. Together, these data indicate that CLASP localizes to the subcellular domain, where microtubule advance into the leading edge is controlled (Lee, 2004).
However, the most surprising feature came from a quantitative analysis of the rates of microtubule extension. For this, CLASP-expressing neurons were grouped into low-expressing neurons and compared to high expressing neurons that still showed some degree of microtubule dynamics. Microtubule advance was found to be significantly slower in growth cones with high CLASP expression. This correlated with a reduced leading edge advance in growth cones displaying high CLASP-GFP expression. Thus, CLASP can impede microtubule growth toward the leading edge and cause the growth cone to slow down. This supports the genetic data that place CLASP downstream of a growth cone repellent (Lee, 2004).
Drosophila oocyte differentiation is preceded by the formation of a polarised 16-cell cyst from a single progenitor stem cell as a result of four rounds of asymmetric mitosis followed by incomplete cytokinesis. The Orbit/Mast microtubule-associated protein is required at several stages in the formation of such polarised 16-cell cysts. In wild-type cysts, the Orbit/Mast protein not only associates with the mitotic spindle and its poles, but also with the central spindle (spindle remnant), ring canal and fusome, suggesting it participates in interactions between these structures. In orbit mutants, the stem cells and their associated fusomes are eventually lost as Orbit/Mast protein is depleted. The mitotic spindles of those cystocytes that do divide are either diminutive or monopolar, and do not make contact with the fusome. Moreover, the spindle remnants and ring canals fail to differentiate correctly in such cells and the structure of the fusome is compromised. The Orbit/Mast protein thus appears to facilitate multiple interactions of the fusome with mitotic spindles and ring canals. This ensures correct growth of the fusome into a branched asymmetrically distributed organelle that is pre-determinative of 16-cell cyst formation and oocyte fate specification. Finally the Orbit/Mast protein is required during mid-oogenesis for the organisation of the polarised microtubule network inside the 16-cell cyst that ensures oocyte differentiation. The localisation of CLIP-190 to such microtubules and to the fusome is dependent upon Orbit/Mast to which it is complexed (Máthé, 2003).
The Orbit/Mast protein facilitates a wide array of cytoskeletal events in both mitotic and interphase cells during Drosophila oogenesis. Failure to provide these functions affects organisation of the stem cell niche, the polarised growth of the fusome, the division of germline cells, specification and maintenance of oocyte fate, and the establishment of the microtubule mediated system for polarised nurse cell-oocyte transport (Máthé, 2003).
As orbit6 females age they ultimately show a complete failure of egg chamber production, reflecting an eventual loss of stem cells from the ovariolar niche. The wild-type ovariolar niche consists of two germline stem cells and the somatic terminal filament and cap cells thought to cooperate in establishing and maintaining stem cell identity. This process requires that stem cells divide asymmetrically along the anteroposterior axis of the germarium such that only their anterior daughter cells inherit cap cell contact and a larger piece of fusome. The posteriorly situated daughter cells that inherit a smaller piece of fusome lose contact with cap cells and differentiate into cytoblasts. The orbit6 phenotype indicates that the gene product is required for stem cell maintenance. One possible explanation offered for this might be that the absence of Orbit/Mast protein leads to stem cell division defects that cause stem cells to lose contact with the cap cells and the terminal filament cells (Máthé, 2003).
The ovaries of younger females do have stem cells that are able to produce egg chambers, although these have reduced numbers of nurse cells. This reflects directly the requirement of Orbit/Mast protein for cell division and/or fusome growth. During mitosis of the germline cells, the Orbit/Mast protein was found to associate with the spindle microtubules and spindle poles; towards the completion of cell division it became concentrated in the spindle remnants, as reported in other cell types (Inoue, 2000; Lemos, 2000). Indeed, mitotically dividing orbit6 cystoblasts show a variety of abnormalities consistent with defects seen in dividing somatic cells of other orbit alleles. These include short bipolar and monopolar spindles, supporting recent observations that the mitotic requirement for Orbit/Mast is to maintain spindle bipolarity (Maiato, 2002). Orbit/Mast protein is also found in the fusome that, in wild-type cells, is always associated with one pole of each mitotic spindle in the cyst of dividing cells. By contrast, the orbit6 spindles never make contact with fusome fragments. Loss of connections between spindles and fusome have been described in mutants for the heavy chain of the minus end directed motor dynein (Dhc64C) and in germline clones of null alleles of Lis1, which encodes the Drosophila homolog of the lissencephaly disease gene, the product of which interacts with dynein. The phenotypes in each case have some similarities to orbit6: the spindles often fail to attach to fusomes, cysts are produced with fewer than 16 cells, and the oocyte is not specified. It has been suggested that these defects are due to interactions between fusome and spindles or interphase microtubules. Although the defects seen in orbit6 could arise in this way, the mitotic defects seen in the mutant give greater emphasis to the necessity to maintain correct interactions between the metaphase spindle and the fusome and suggest that Orbit/Mast protein may have a direct role in ensuring such connections (Máthé, 2003).
The fusome provides a physical basis to ensure asymmetry of the developing cyst that is a precondition for cyst polarisation and oocyte specification. Fusome material does develop in the cysts of younger orbit6 females, but tends to remain as separate fragments. The failure of correct fusome development is most likely to be the underlying cause behind the failure to specify the oocyte in orbit6 mutants. The data suggest that not only is Orbit/Mast required for interactions of the fusome with spindles, but it is also needed for its subsequent interactions with spindle remnants and the ring canals. Fusome material accumulates in the vicinity of the spindle remnants in wild-type cysts. These spindle remnants have been suggested to help restrict constriction of the cleavage furrows and so guide fusome growth by supporting the migration of fusome plugs and their ring canals towards the pre-existing fusome. The absence of spindle remnants in the mutant orbit6 cysts may therefore also contribute to the lack of fusome growth. One possibility is that the transfer of Orbit/Mast protein from the degenerating spindle remnants to the fusome plugs inside the ring canals themselves may facilitate migration of fusome plugs along interphase microtubule bundles to fuse with pre-existing fusome. This would ensure the regular and polarised growth of the fusome (Máthé, 2003).
The defects observed in ring canal formation in orbit6 mutants are almost certainly secondary to the failure of the fusome to develop correctly. The central spindle (spindle remnant) late in mitosis is important to arrest closure of the cleavage furrows so they may be transformed into ring canals. However, the ring canals continue to differentiate even after completion of cystocyte divisions. Anillin, glycoprotein D-mucin, Orbit/Mast and Pav-KLP are the earliest known proteins to associate with the ring canals during germline cell divisions. Upon completion of these divisions, Anillin and Orbit/Mast disappear from the ring canals, while glycoprotein D-mucin and Pav-KLP are joined by Filamin, HtsRC and Kelch. The ring canals that do form in younger orbit6 females are occluded by Filamin, F-actin and HtsRC. A failure to recruit Kelch may be a secondary consequence of the formation of such aberrant structures. Ring canal occlusion could be the result of the irregular structure of the spindle remnant in dividing orbit6 germline cysts and the failure to plug the newly formed ring canal with fusome components. Mutant egg chambers from older females show stronger defects; often they contain irregular structures that contained HtsRC, Filamin and F-actin but do not resemble ring canals. This gradient of phenotype along the ovariole may reflect once again the exhaustion of a diminished pool of Orbit/Mast protein as development proceeds (Máthé, 2003).
Molecules found in the fusome are normally found in the cortical cytoskeleton of most other cell types. Thus, the involvement of Orbit/Mast in the interactions of microtubules with the fusome may be akin to the interphase functions ascribed to CLASP, its counterpart in mammalian cells (Akhmanova, 2001). Interactions between the CLASP, CLIP-170, EB1 and APC proteins have been proposed to mediate the crosstalk between cellular structural elements, particularly actin filaments, microtubules and the plasma membrane. The notion of Orbit/Mast having an equivalent role in the Drosophila egg chamber is supported by the finding that the Orbit/Mast protein exists in a complex with CLIP-190, the counterpart of CLIP-170. The two proteins can be co-immunoprecipitated and overlap considerably in their pattern of subcellular localisation throughout oogenesis (Máthé, 2003).
A further interphase role for the Orbit/Mast protein in oogenesis would seem to be in facilitating the organisation of the polarised network of microtubules essential for transport of mRNAs and proteins from the nurse cells to the growing oocyte at stages 1 to 7. The minus ends of microtubules in this network are nucleated by a unique MTOC situated at the posterior of the oocyte. The Orbit/Mast protein is present in punctate bodies along these microtubules and accumulates at this MTOC. Its accumulation at the MTOC requires functional microtubules, since it fails to accumulate in females treated with a microtubule depolymerising drug. This is one aspect of its localisation in which it forsakes its partner CLIP-190, which (although present in punctate bodies associated with the microtubule network) is absent from the MTOC. The mechanisms that regulate the association of these two proteins with respect to the polarity of microtubules are likely to be significant in the full understanding of their biological roles. The polarised microtubule network is disrupted and the MTOC is reduced or absent from egg chambers of orbit6 females. Once again this phenotype is age dependent: younger mutant females feature only irregular MT bundles and diminutive irregularly placed MTOCs whereas the bundles and MTOC are totally absent in egg chambers of older females. The localisation of CLIP-190 to the fusome and to the polarised microtubule array is also prevented in the orbit6 mutant, suggesting that it requires complexing to the Orbit/Mast protein to achieve this distribution (Máthé, 2003).
Taken together, these observations strongly argue that the Orbit/Mast protein is required for major polarisation events in wild-type egg chambers: the establishment of the polarised fusome that ultimately enables a single cell within the 16-cell cyst to be specified as the oocyte and in the formation of the polarised MT network in mid-oogenesis. Further studies are needed to determine the precise mechanism whereby the Orbit/Mast protein participates in the organisation of the mitotic spindle, fusome and ring canals, on the one hand, and in the organisation of polarised arrays of interphase microtubules, on the other; and to determine whether the other Drosophila orthologs that are members of the CLASP complex are also involved in these processes (Máthé, 2003).
Regulation of cytoskeletal structure and dynamics is essential for multiple aspects of cellular behavior, yet there is much to learn about the molecular machinery underlying the coordination between the cytoskeleton and its effector systems. One group of proteins that regulate microtubule behavior and its interaction with other cellular components, such as actin-regulatory proteins and transport machinery, is the plus-end tracking proteins (MT+TIPs). In particular, evidence suggests that the MT+TIP, CLASP (Chromosome bows), may play a pivotal role in the coordination of microtubules with other cellular structures in multiple contexts, although the molecular mechanism by which it functions is still largely unknown. To gain deeper insight into the functional partners of CLASP, parallel genetic and proteome-wide screens for CLASP interactors were performed in Drosophila. 36 genetic modifiers and 179 candidate physical interactors were identified, including 13 that were identified in both data sets. Grouping interactors according to functional classifications revealed several categories, including cytoskeletal components, signaling proteins, and translation/RNA regulators. The initial investigation focused on the MT+TIP Minispindles (Msps), identified among the cytoskeletal effectors in both genetic and proteomic screens. This study reports that Msps is a strong modifier of CLASP and Abl in the retina. Moreover, Msps functions during axon guidance and antagonizes both CLASP and Abl activity. The data suggest a model in which CLASP and Msps converge in an antagonistic balance in the Abl signaling pathway (Lowery, 2010).
The in vivo functions of cytoskeletal effector and regulatory proteins have been studied very effectively in Drosophila, with particular success at the earliest stages of embryonic development prior to zygotic gene expression, when depletion of maternal stores of such proteins often results in disruption of mitosis, cellularization, or other aspects of cell biology. However, the functions of cytoskeletal effectors at late stages of development are often obscured by early embryonic functions. In this regard, the existence of maternal stores of some key effectors has been helpful for analysis of late events in nervous system development, such as axonal and dendritic patterning, because such maternal supplies of protein are sometimes exhausted only at late stages when axons and dendrites emerge. However, zygotic mutations in many key cytoskeletal components disrupt early stages, making screens based on neuroanatomical phenotypes problematic. For this reason, genetic interaction screens were used to explore the network of cytoskeletal regulators linked to key guidance signaling molecules as a means of identifying candidates for deeper analysis during axonal development. These screens for modifiers of Abl kinase phenotypes led to the identification of CLASP as an effector essential for accurate growth cone navigation. By using CLASP as a starting point for a new generation of screens, new functional categories and individual players of the CLASP interactome were identified, including cytoskeletal components, signaling proteins, and translation/RNA regulators. In addition, a microtubule regulatory protein (the MT+TIP Msps) not previously associated with axonal pathfinding decisions was identified (Lowery, 2010).
To build functional neural networks, axonal growth cones must accurately interpret and translate multiple guidance cues into directional movement by coordinating both microtubule and F-actin networks. There appears to be significant interplay between the two cytoskeletal components, but a sophisticated understanding of the signaling and effector mechanisms by which both systems are coordinated in response to guidance cues has not yet been obtained. The Abl tyrosine kinase is one of the few known signaling molecules shown to transduce guidance cue signals to both actin and MT networks, although far less is known regarding how it regulates MTs. This work, and that of others, suggests that CLASP may be an important player in the MT-actin crosstalk machinery (Lowery, 2010).
The largest thematic group of CLASP-interacting genes identified is the actin-binding proteins, including Shot, Zip, Capu, Pnut, Jar, Bif, Kst, and the uncharacterized CG13366 (ortholog of calponin-homology domain containing CYTSA/B). These are all known actin-associated factors that are also predicted to bind to MTs, and their presence in the screen points to a role for CLASP in mediating actin dynamics in coordination with the MT network. This supports previous observations that vertebrate CLASPs may function as actin-MT crosslinkers. CLASPs possess actin-binding activity (Tsvetkov, 2007), and CLASP-decorated MT tips track along actin filament bundles in the growth cone peripheral domain. Moreover, CLASP was recently shown to bind to the actin-binding protein, IQGAP1, and phosphorylation of CLASP controls linkage of MTs to actin through IQGAP1 for cell migration (Watanabe, 2009). From the current study, it appears that CLASP may have numerous other effector proteins that can modulate its interaction with the actin network. IQGAPs have not been found in Drosophila, and so, it is speculated that the novel interactors identified, as well as others, may allow CLASP to link MTs and actin in different contexts (Lowery, 2010).
The novel CLASP MT+TIP interactor that were identified, Msps, emerged from the screens as a high priority for future analysis. Msps interacts with CLASP in both the genetic and proteomic screens, and it antagonizes CLASP and Abl signaling. The antagonism seen between Msps and CLASP in the Drosophila retina is consistent with cell culture studies, which have shown that CLASP regulates MT dynamics by specifically promoting the pause state (Sousa, 2007) whereas Msps-family proteins function as MT antipause factors (Brittle, 2005). More specifically, CLASPs have MT-stabilizing effects, and depleting cells of CLASP protein results in highly dynamic, constantly growing or shrinking MTs (Sousa, 2007). Alternatively, Msps family members can have the opposite effect (Popov. 2003), catalyzing the addition and removal of multiple tubulin dimers at MT plus-ends (Brouhard, 2008), and depletion of Msps results in a dramatic increase in MT pausing with little or no growth (Brittle, 2005). These opposite effects on MT behavior in cell culture studies suggest reciprocal functions in the regulation of MT dynamics in vivo, and Msps could thus be a component of the Abl signaling pathway that provides an antagonistic counterbalance to CLASP in regulating the growth cone cytoskeletal output downstream of guidance cues (Lowery, 2010).
In the context of CNS, it seems likely that CLASP and Msps drive axon guidance decisions through reciprocal regulation of growth cone turning toward or away from the source of axon guidance factors at the midline. Accurate navigation of both ipsilateral and contralateral axon pathways requires a combination of cues, to regulate both midline crossing behavior and also the stereotyped positions of longitudinal axon tracts. The lateral specification model proposes that for longitudinal axons to find and maintain a correct trajectory at a specific distance from the midline, they must reach a balance of turning responses to the attractive Netrins and repellent Slit secreted by midline glia. Perhaps this balance requires antagonism of Msps and CLASP downstream of Abl, such that reduction of either protein would bias the growth cone or reduce the fidelity of the overall navigation process. Abl has been shown to mediate both Slit and Netrin activity, thus providing a potential point of integration (Lowery, 2010).
While it is anticipated that CLASP and Msps will influence the directionality of growth cone advance in response to guidance cues, the cellular mechanism by which these two effectors guide axons is not yet known. Reciprocal control over MT advance toward the growth cone peripheral domain could account for the effects of CLASP and Msps. However, there are alternatives. For example, reciprocal modulation of growth cone cell adhesion by Msps and CLASP might underlie the two phenotypes observed in the different mutants. Reduction of adhesion has already been shown to be key in the midline repulsive response to Robo, whereas an increase in adhesion has long been known to be vital for fasciculation. Interestingly, the Abl kinase that interacts with both Msps and CLASP was also implicated in Robo-dependent modulation of cell adhesion. If CLASP were to play a role in Robo-mediated suppression of adhesion, then the ectopic midline crossing that occurs in CLASP LOF mutants could be explained by an increase in growth cone adhesion toward the midline, which is exacerbated when Msps is overexpressed. Consistently, the fasciculation morphology defects that occur in msps LOF (and are exacerbated by CLASP GOF) could be explained if the role of Msps is to promote adhesion. Although the effects of CLASP-family proteins on cell adhesion have not been directly measured, studies in nonneuronal contexts suggest that CLASP helps to drive MT-cortical interactions, which would presumably promote, not suppress, adhesion (Lowery, 2010).
In conclusion, this is the first study demonstrating that Msps functions during axon guidance. Numerous studies have analyzed its role in the regulation of MT stability in several systems including the mitotic spindle and in centrosomes, but its potential role(s) in the nervous system has never been previously addressed. In fact, the growth cone functions of most MT+TIPs are unknown; however, previous discoveries that MT+TIPs CLASP and APC, and now Msps, are important for axon guidance demonstrates that the MT+TIPs are an exciting class of guidance effectors worthy of further exploration and understanding (Lowery, 2010).
A Drosophila gene, orbit, encodes a conserved 165-kD microtubule-associated protein (MAP) with GTP binding motifs. Hypomorphic mutations in orbit lead to a maternal effect resulting in branched and bent mitotic spindles in the syncytial embryo. In the larval central nervous system, such mutants have an elevated mitotic index with some mitotic cells showing an increase in ploidy. Amorphic alleles show late lethality and greater frequencies of hyperploid mitotic cells. The presence of cells in the hypomorphic mutant in which the chromosomes can be arranged, either in a circular metaphase or an anaphase-like configuration on monopolar spindles, suggests that polyploidy arises through spindle and chromosome segregation defects rather than defects in cytokinesis. A role for the Orbit protein in regulating microtubule behavior in mitosis is suggested by its association with microtubules throughout the spindle at all mitotic stages, by its copurification with microtubules from embryonic extracts, and by the finding that the Orbit protein directly binds to MAP-free microtubules in a GTP-dependent manner (Inoue, 2000).
The original orbit mutation was identified within a subset of a collection of P element-induced mutants that showed maternal-effect lethality. Homozygous orbit1 mutants are fully viable, except that their development is delayed by a number of days in crowded cultures. However, homozygous orbit1 females lay fewer than 10% of the eggs of wild-type females, the numbers decreasing markedly as mutant females age. Consistently, ovaries of orbit1 females show degeneration and contain fewer egg chambers than wild-type. Approximately one third of the orbit1-derived eggs possessed no nuclei, suggesting there had been defective premeiotic and/or meiotic divisions, and of the remainder, >90% showed perturbation of the uniform distribution of the nuclei. 10%-20% of embryos derived from orbit1 females cellularized, at least partially, but <1% hatched. Homozygous males for orbit1 mutation were also sterile (Inoue, 2000).
To better understand how the abnormal nuclear density might arise in orbit1-derived embryos, the distribution and organization of centrosomes and spindle microtubules was examined during the syncytial nuclear division cycle. Whereas in wild-type embryos there is a regular distribution of mitotic spindles at metaphase, in orbit1-derived embryos at a similar stage there are regions devoid of nuclei that contain free centrosomes that nucleate asters of microtubules. Moreover, additional centrosomes appear to become incorporated into spindles to form multipolar structures. Free centrosomes could also be observed in fields of anaphase figures from the mutant in which the spindles were frequently excessively curved, bent, and sometimes wavy. This defect appeared accentuated at telophase where the midbodies could be disoriented so that they aligned at 90° rather than 180° to each other. When syncytial orbit1-derived embryos were stained with Hoechst to reveal DNA, nuclei that were more brightly stained than their neighbors were frequently observed. These appear to contain more than a diploid amount of DNA, and could either be the outcome of failed nuclear separation or the fusion of nuclei. In addition, nuclei connected by thin chromatin bridges were occasionally seen, suggesting failure of chromatid disjunction (Inoue, 2000).
The rapidity of the nuclear division cycles in syncytial embryos, and the absence of certain checkpoint controls make it difficult to observe primary defects in cell cycle mutants. The availability of additional orbit alleles showing late larval lethality therefore prompted an investigation of the effects of these mutations on progression of mitosis in dividing somatic cells. Mitotic defects were found in cells of the larval CNS not only in the amorphic alleles generated by P element mobilization, but also in the original orbit1 mutant. In contrast to wild-type cells where hyperploid cells are never seen, ~6% of total metaphase cells in squashed preparations of the larval CNS from the orbit1 homozygotes contained more than a diploid complement of chromosomes. The overall mitotic index in the larval CNS was almost three times higher than wild-type, and moreover, the proportion of diploid cells in metaphase to anaphase was two times higher in orbit1 than in wild-type, indicative of a delay in the passage though this mitotic transition. The majority of the polyploid figures and 18% of the diploid figures in orbit1 homozygotes contained hypercondensed chromosomes, suggesting that these cells had been delayed in mitosis for a significant period of time. In orbit1, the level of hyperploidy of most cells did not exceed 8N. Such hyperploidy could arise by a failure of either chromosome segregation or cytokinesis. However, the metaphase arrest or delay would seem to indicate the former possibility as being more likely. An early mitotic defect is also suggested by the observation of a low frequency of circular mitotic figures similar to those seen in mgr (merry-go-round) and aur mutants. These have all the major chromosomes arranged in a circle with their centromeres inward and arms oriented toward the periphery and the fourth chromosomes at the center, an arrangement that is extremely rare in squashed preparations of wild-type brains. Monopolar anaphase-like were also observed, in which chromatids appear as if pulled towards a single pole (Inoue, 2000).
In orbit1/Df(3L)Pc-9a hemizygotes, the proportion of polyploid cells, the proportion of diploid metaphase figures containing hyper-condensed chromosomes, and the ratio of metaphase to anaphase figures all increased, confirming the hypomorphic nature of this allele. Heterozygotes of orbit1 and each of the three mutations, Df(3L)orbit2, orbit3, and Df(3L)orbit4, showed comparable phenotypes to orbit1/Df(3L)Pc-9a, consistent with the amorphic nature of these three lethal alleles resulting from sequence deletions. The proportion of the circular metaphase figures in hemizygotes for orbit1 or trans-heterozygotes between orbit1 and the amorphic alleles did not increase significantly compared with that in orbit1 homozygotes. The loss of orbit function was examined in orbit3/Df(3L)Pc-9a or orbit3/Df(3L)orbit2. The third instar larvae of these mutants were lacking imaginal discs and had a small larval CNS characteristic of many cell division cycle mutants. They exhibited an extremely high proportion of polyploid cells of between 80% to 90% of the total metaphase figures. The extent of polyploidy was also increased in these mutants with >30% possessing greater than an 8N chromosome complement. It appears that these highly polyploid cells are the result of multiple cell cycles in which chromosome segregation has failed (Inoue, 2000).
In contrast to the bipolar spindles organized by two fully separated centrosomes observed in all wild-type cells, ~10% of mitotic cells observed in the larval CNS of the hypomorphic mutant orbit1 contained a polyploid set of chromosomes associated with spindles that were frequently multipolar. Consistent with the quantitation of orcein-stained figures in squashed preparations, some 3%-5% of mitotic cells contained monopolar mitotic spindles. In some cases, these had a hemispindle-like structure in which chromosomes appeared to be pulled towards a single pole. In others, condensed chromosomes were arranged around a single centrosome on the same plane. These figures appear to correspond to the circular mitotic figures presented in squashed preparations of larval CNS. The finding of monopolar spindles, together with cells that have a reduced number of centrosomal bodies relative to their chromosome complement, suggests that a primary defect in orbit mutants might be a failure of spindle pole separation that ultimately leads to polyploidy (Inoue, 2000).
The mutant phenotype of orbit is suggestive of a role for the wild-type gene in the functioning of the mitotic spindle consistent with the gene product being a novel MAP. This discovery helps overcome the difficulty in interpreting mitotic phenotypes in syncytial embryos derived from homozygous mutant females. Such difficulties arise since syncytial embryos lack certain checkpoints and so aspects of mitotic cycles can continue even though other steps are blocked. This is reflected by the finding of free centrosomes in orbit1-derived embryos that appear to be undergoing autonomous duplication cycles, as seen in many other mitotic mutants. Maternal-effect mutations leading to mitotic defects are often hypomorphic, and have some residual function that allow the homozygous mothers to survive to adulthood partly assisted by a supply of wild-type gene product from the heterozygous grandmother. orbit1 is no exception to this rule, and indeed it proved possible to make amorphic alleles that show larval lethality by remobilization of the P element responsible for the original mutation. Nevertheless, the characteristic spindle defects of two types seen in orbit1-derived embryos reflect the specific effect of diminution of the levels of Orbit protein. The branched spindles could either be an immediate consequence of failure in centrosome duplication or separation, or they could arise by capture of a free centrosome by an otherwise bipolar spindle. In either case, these defects, together with the high proportion of the spindles with wavy or bent arrays of microtubules, indicate a role for the Orbit protein in regulating function of spindle microtubules. Branched spindle defects are also seen in aurora-derived embryos thought to be defective in aspects of centrosome separation, and as with orbit1, are often associated with the generation of what appear to be tetraploid nuclei in the syncytial blastoderm. Such nuclei could arise either as a consequence of the failure of chromosome segregation, or a refusion of sister chromatids or sister nuclei after segregation. The finding of wavy and bent spindle microtubules, however, is not seen in aur-derived embryos and resembles more the maternal-effect phenotype described for certain alleles of abnormal spindle. Taken together, the different aspects of the maternal-effect phenotype suggest a primary defect in spindle microtubule function leading to failure of chromosome segregation (Inoue, 2000).
Defective spindle microtubule function is also evident in the developing larval central nervous system of orbit1 mutants. A high frequency of cells in a metaphase-like state suggests that the spindle integrity checkpoint has been activated to delay progression through mitosis. The high degree of chromosome condensation provides further evidence that the cells have been arrested at this point for some time, during which there has been continued activity of p34cdc2. There are two characteristic features of the arrested cells in the orbit1 mutant; a low frequency of monopolar mitotic structures and also polyploid cells. The proportion of polyploid cells increases when the orbit1 mutation is hemizygous, indicative of its hypomorphic nature. In the amorphic mutant combinations, monopolar figures are no longer seen and virtually all cells become polyploid and at much greater levels. Polyploid cells could arise either through a defect in chromosome segregation followed by exit from mitosis, and subsequent reentry into the next mitotic cycle, or alternatively, there can be a failure of cytokinesis. The findings of a high mitotic index with very few anaphases, and the presence of monopolar figures, strongly suggests that the polyploidy arises as a consequence of spindle defects leading to a failure of chromosome segregation. Of course, this would not preclude some function for the Orbit protein in the late mitotic spindle, the correct structure of which is essential for cytokinesis to take place. However, the low incidence of anaphase figures in orbit mutants suggests that mitotic events rarely proceed to this stage (Inoue, 2000).
The high levels of polyploidy attained in cells of the amorphic orbit mutants indicate that they have gone through repeated cell cycles without division, and preclude analysis of the primary mutant defect. The hypomorphic orbit1 mutant, in contrast, allows a glimpse of those aspects of mitosis that are most sensitive to diminished Orbit function. The observation of a low frequency of monopolar spindle structures suggests that Orbit assists in promoting the correct separation of centrosomes to form a bipolar spindle structure. However, there would seem to be other requirements for the Orbit protein in the spindle since bipolar spindles do form, which then appear to undergo spindle checkpoint arrest at metaphase. Indeed, the centrosome separation defect may be secondary to a spindle microtubule function. In this respect, orbit mutants differ from mgr or aur, which appear to have a more direct role in centrosome separation. Not only is the frequency of monopolar mitotic figures lower in orbit than in merry-go-round or aurora, but also monopolar structures are not seen in amorphic orbit mutants, whereas they increase in frequency in amorphic aurora mutants. This suggests a direct role for the Aurora protein kinase in centrosome separation, such that in its absence the mitotic cycle is definitively arrested at this point. In contrast, the decrease in mitotic index and accompanying increase in levels of polyploidy in amorphic orbit mutants is indicative of cells continually delayed and repeatedly leaking through the spindle integrity checkpoint. The structure of the monopolar figures also differs between these mutants. In mgr and aur, the chromosomes are invariably arranged in a circle in a metaphase-like state as if under tension with their centromeres pulled towards (but always at some distance from) the center of the circle, and the chromosome arms pulled out towards the periphery. Similar figures are seen in orbit1, but in addition, there are anaphase-like figures in which the centromeres appear to have been pulled into the immediate vicinity of a single pole. These cytological phenotypes more closely resemble those in the mutants for the kinesin-like protein, KLP61F, first thought to be required for centrosome separation at prophase, but then shown by antibody injection experiments to be required for maintenance of spindle bipolarity. The Orbit protein appears to be localized throughout the mitotic spindle like the KLP61F protein, although at the EM level it is apparent that KLP61F is not uniformly distributed (Inoue, 2000).
Orbit protein is associated with all spindle and astral microtubules at all stages of the mitotic cycle, and microtubules from embryo cytoplasm copurify with Orbit protein attached to them. The lower ratio of Orbit:tubulin in the microtubule pellet fraction, compared with a crude extract could suggest that not all of the Orbit protein is bound to microtubules. Alternatively, the affinity of Orbit for the taxol polymerized microtubules used in these experiments could be lower than for naturally polymerized microtubules, a possibility currently under investigation. The primary sequence of the Orbit protein reveals it to be a basic protein, a characteristic of MAPs. Moreover, within these highly basic regions are motifs that strongly resemble sequences present in the vertebrate and yeast MAPs, MAP4 and Stu1p. Three distinctive features in the microtubule-binding domains of MAP2, tau, and MAP4 have been described. Polypeptides comprising these different domains cause microtubules assembled in vitro to adopt different shapes. Similarly, microtubules assembled in the presence of the individual neuronal MAPs, MAP1A, MAP1B, and MAP2 have also been shown to adopt a variety of shapes from 'short and straight' to 'long and bendy'. Thus, the bending of spindle shape seen in orbit1 embryos may be indicative of requirement for the Orbit protein to confer a certain shape to the spindle microtubules (Inoue, 2000).
Many of the first MAPs to be characterized were obtained from preparations of tubulin from mammalian brain, and are likely to have their primary function in the neuronal cytoskeleton. Nevertheless, it is now appreciated that some of these proteins are expressed in other tissues in which there is cell proliferation. Phosphorylation of the Xenopus homolog of MAP4 by both p34cdc2 and mitogen-activated-protein kinases appears important for its microtubule-binding and stabilizing properties and for chromosome movement during anaphase A. Similarly, Stathmin/Op18 is a protein that interacts with tubulin to inhibit microtubule polymerization. Overexpression of its nonphosphorylatable forms prevents mitotic spindle assembly in tissue culture cells , whereas its phosphorylated form stimulates microtubule growth around chromosomes. In this context, the conserved p34cdc2 sites in Orbit may well play a role in regulating its function. Moreover, the abundance of serine residues within two regions of the protein may be indicative of sites for phosphorylation by other mitotic kinases, such as Polo or Aurora, whose consensus sites are not yet known (Inoue, 2000).
Other MAPs can act through destabilizing the polymers, for example the Kin I kinesins, and others, such as katanin, can actually sever the microtubules. Three Xenopus MAPs that each localize to the spindle have different effects on microtubule dynamics: XMAP215 promotes microtubule growth, XMAP230 decreases the catastrophe frequency, and XMAP310 increases the rescue frequency. It is of considerable future interest to determine whether the Orbit protein has any effect upon these parameters of microtubule dynamics (Inoue, 2000).
Through mutational analysis in Drosophila, the gene multiple asters (mast), has been identified that encodes a 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by an irregular number of microtubule-organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologs have been identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein-Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule-binding assays indicate that Mast is a microtubule-associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle (Lemos, 2000).
Genetic analysis of the mast locus shows that this protein is required for the organization of the mitotic spindle. Mutant neuroblasts show highly abnormal mitotic figures, rarely organize bipolar spindles and most of them contain one or more MTOCs of irregular size and shape. Detailed analysis of the abnormal MTOCs indicates that these are composed mostly of multiple CNN-positive ring-like structures that are present in all normal centrosomes. Therefore, it is unlikely that mutations in mast cause an abnormal organization of the centrosome itself. Furthermore, the abnormal MTOCs observed in mast mutant cells contain many CNN-positive ring-like structures, suggesting that centrosome replication is not affected. One possible interpretation of the data could be that in mutant cells, centrosomes do segregate but collapse at a later stage, forming a monopolar configuration as was shown for mutations in KLP61F. However, in strong mutant alleles, multiple MTOCs of irregular size are associated with MT asters that are found dispersed throughout the cell. In this context, mutations in mast display a phenotype similar to that of C. elegans zyg-9, which shows numerous cytoplasmic clusters of short MTs during meiosis. However, ZYG-9 is thought to be required for the organization of long MTs during meiosis, and mutations in mast do not appear to cause a significant reduction in MT length. Nevertheless, the morphology of MTs in mast mutant cells is not normal, appearing to be much more irregular in shape. Furthermore, disruption of STU1 in S. cerevisiae causes severe defects in spindle assembly (Pasqualone, 1994). Accordingly, the highly abnormal pattern of centrosome segregation observed as a result of mutations in mast could be due to abnormal MT organization (Lemos, 2000).
If Mast is essential for normal centrosome segregation and bipolar spindles are rarely observed, it might be expected that the spindle checkpoint would prevent these cells from advancing into a new cycle of proliferation. However, despite the increase in mitotic index, mutations in mast cause the formation of highly polyploid cells. Mutant cells respond to the spindle checkpoint when arrested with colchicine in prometaphase, since premature sister chromatid separation is never observed. Furthermore, mutant cells with abnormal spindle morphology and highly condensed chromosomes show strong accumulation of the spindle checkpoint protein Bub1. This staining pattern is similar to that observed in chromosomes of cells arrested in prometaphase after MT depolymerization, suggesting that in mast mutant cells the interactions that occur between MTs and chromosomes are unable to inactivate the spindle checkpoint, leading to a prolonged prometaphase arrest. However, since highly polyploid cells are formed, these cells must have undergone multiple cell cycles in the absence of chromosome segregation and cytokinesis. Therefore, it is most likely that after some time, these cells adapt; they either become insensitive or override the spindle checkpoint and progress into a new cycle of proliferation. Indeed, the length of time that different cell types remain in M phase in the presence of microtubule inhibitors varies widely (Lemos, 2000).
Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that is required for the organization of the mitotic spindle. Evidence is provided that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics are suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity (Maiato, 2002).
These studies provide strong evidence suggesting that MAST/Orbit is required for microtubule plus ends to establish a functional attachment to kinetochores. In the absence of MAST/Orbit, chromosome congression does not take place and kinetochores either do not show a clear end-on attachment or appear to bind through lateral interactions. Most surprising is the monopolar configuration in MAST-depleted cells where chromosomes are found mostly localized close to the center of the aster. If, in MAST-depleted cells, the microtubule-kinetochore interaction is compromised, it would be expected that the action of chromokinesins and even passive impacts by elongating microtubules would push the chromosomes to the periphery of the asters (the 'polar wind'). This suggests that the localization of chromosomes to the interior of asters is likely to involve an active, rather than a passive, process that can be explained by at least three different models. MAST/Orbit could be required for kinetochores to hold on to dynamic microtubules. If it were true that in cells lacking MAST/Orbit, kinetochores could not hold onto shortening microtubules, then every time a kinetochore fiber would begin to shorten, it would be released by its kinetochore. That kinetochore would then have to make an initial encounter with microtubules all over again through lateral interactions that could be mediated by cytoplasmic dynein, as it is during prometaphase, and the chromosome would be expected to exhibit rapid poleward movement. Consistent with this model, levels of kinetochore-associated dynein and ZW10 remained abnormally elevated in the absence of MAST. A second interpretation of these results is that in the absence of MAST/Orbit, chromosomes attach to microtubules that can shorten but cannot regrow so that chromosomes would end up mostly in the region close to the center of the aster, suggesting that MAST/Orbit could have a role in promoting microtubule plus end stability. Indeed, the human homologs, CLASPs, have been shown to promote plus end microtubule stabilization in interphase (Akhmanova, 2001). However, MAST/Orbit does not appear to affect the ability of all microtubules to elongate, because in MAST-depleted cells, there is no obvious effect upon growth of astral microtubules. Therefore, if MAST/Orbit has a role in microtubule dynamics, kinetochore microtubules are particularly sensitive. Finally, it is also possible that in MAST-depleted cells, kinetochores attach to microtubules, but minus end-directed motility of the kinetochore dominates so that the movement of chromosomes toward the poles prevails over the polar wind, pushing the chromosomes toward the plus ends of microtubules. It could be that kinetochore-associated MAST (Lemos, 2000) is required for the binding of essential kinesins with plus end-directed motility. A putative candidate could be CENP-E, a plus end-directed motor that has been shown to localize to the fibrous corona of the kinetochore and be required for stable, bioriented attachment of chromosomes to spindle microtubules. Surprisingly, depletion of CENP-E by microinjection also causes misaligned chromosomes to be sequestered very close to the spindle poles (Maiato, 2002 and references therein).
Although, at present, the data presented here does not allow for distinguishing between these interpretations, the first two models are favored because they could readily explain the results obtained after taxol treatment of MAST-depleted cells. Suppressing microtubule dynamics after depletion of MAST/Orbit causes the formation of monopolar cells, with most kinetochores associated to the plus ends of microtubule bundles. MAST/Orbit could be part of a protein complex required to hold on to the plus ends of dynamic microtubules. Alternatively, MAST/Orbit could be required for microtubule plus end stabilization. Thus, in the absence of MAST/Orbit, kinetochores attach to microtubules, and facilitating their stabilization could allow the formation of a kinetochore fiber and its growth. Consistent with this interpretation, it has been shown that the fission yeast ortholog of CLIP-170, tip1p, is an anticatastrophe factor (Brunner, 2000). Thus, it is possible that the defects observed after loss or mutation of MAST could be due to an effect on D-CLIP-190 (the Drosophila homolog of mammalian CLIP-170). However, it has been shown that the localization of D-CLIP-190 to kinetochores is normal in the absence of MAST. Nevertheless, it cannot be excluded that MAST could have a dual role affecting both kinetochore attachment and microtubule stability, as has been recently described for Bik1 (Lin, 2001), the budding yeast homolog of CLIP-170 (Maiato, 2002).
In vivo analysis of mast5 mutant embryos demonstrates that MAST becomes essential for the maintenance of a bipolar spindle during metaphase; in its absence, the spindle collapses and centrosomes slide toward the metaphase plate forming monopolar spindles. STU1p, the putative MAST ortholog in S. cerevisiae, is also essential for spindle stability (Pasqualone, 1994). It is now well accepted that a balance between plus end- and minus end-directed motors is essential for the maintenance of a bipolar spindle through metaphase and also for its elongation during anaphase. This has been best studied in S. cerevisiae, where imbalances between the Cin8p/Kip1p and Kar3p kinesins cause rapid collapse of the bipolar spindle. Thus, it is possible that MAST is required for the stable association of one or more kinesins with the microtubules and that in the absence of this kinesin, the balance of power is perturbed at metaphase. Indeed, the collapse of the spindle seen after depletion of MAST is very similar to the effect caused by depletion of the Drosophila bipolar kinesin-like KLP61F. The presence of MAST in the midzone (Lemos, 2000) suggests that MAST may also be acting by cross-linking interpolar microtubules and so stabilizing the spindle. It is also possible that depletion of MAST perturbs the distribution or function of other MAPs that are required for formation of a functional bipolar spindle. These could include the chromosomal passengers INCENP and aurora-B. In fact, aberrant behavior of these proteins is observed in anaphase-like cells after depletion of MAST by dsRNAi (Maiato, 2002).
Although depletion of MAST function by dsRNAi causes most cells to arrest in mitosis with monopolar spindles, after long incubation periods, a proportion of cells is seen to adopt an anaphase-like configuration with paired sister chromatids migrating to the poles. These cells are cyclin B positive, retain BubR1 at the kinetochores, and also appear to undergo cytokinesis with an actin contractile ring. A large proportion of these cells progress into further cycles of DNA replication and become polyploid. Because S2 cells have a functional spindle checkpoint, as shown by their ability to arrest in mitosis in the presence of colchicine and also by their initial accumulation in mitosis after MAST depletion, it is considered likely that MAST-depleted cells bypass the checkpoint and exit mitosis (Maiato, 2002).
Orbit/MAST was identified as a candidate partner of Abl in a screen outside of the embryo where a requirement for Abl in axonogenesis was defined (Wills, 1999a). In a retinal screen, overexpression of orbit/MAST enhanced the AblGOF phenotype, suggesting that these two proteins cooperate in vivo. However, validation of the screen required analysis of mutations in orbit/MAST (Lee, 2004).
Orbit/MAST was initially identified as a maternal effect lethal locus with defects in mitotic spindle and chromosome morphology (Fedorova, 1997; Inoue, 2000 and Lemos, 2000); however, zygotic mutants display no defects in cell division, presumably due to maternal stores of the protein required for oogenesis (Maiato, 2002). Independent LOF alleles were examined for zygotic phenotypes. Axon fascicles that are restricted to either side of the central nervous system (CNS) midline by Slit signaling can be visualized at stage 17 with anti-Fasciclin II (FasII, Mab1D4). In late-stage wild-type embryos (stage 17), FasII is excluded from the midline. However, in orbit/MAST mutants, ectopic midline crossing was detected, primarily by the midline-proximal MP1 axon pathway. This phenotype is qualitatively identical to that seen in Abl zygotic mutants (Wills, 2002 and Hsouna, 2003; note that loss of maternal and zygotic Abl generates catastrophic axonal defects, underlining Abl's central role in axonal development; Grevengoed, 2001). Since the exclusion of FasII from axon commissures reflects a redistribution of protein that could be dependent on Orbit/MAST, it was important to confirm the guidance defects with an alternative marker. Using a Tau-LacZ fusion protein under control of an Apterous promotor expressed in two medial ipsilateral axons that never cross the midline, frequent ectopic crossing of these axons was found in orbit/MAST mutants (Lee, 2004).
In order to rule out the possibility that axonal defects in orbit/MAST alleles result from some early failure in cell division or fate acquisition in the CNS, these homozygous mutants were stained with markers of neuronal cell fate. The number and position of neurons appeared to be normal even in the strongest orbit/MAST alleles. The fate of the midline glia that secrete Slit was examined, but no abnormalities were detected. To prove that the orbit/MAST axon defects represent a late, CNS-specific function of the gene, UAS-orbit(+) was expressed in mutant backgrounds under the control of postmitotic, neuron-specific GAL4 (elav-GAL4 and 1407-GAL4). Quantification of ectopic midline crossing in independent orbit/MAST mutants revealed an allelic series of guidance defects whose penetrance was consistent with the perdurance of some maternal protein. However, two independent transgenes successfully rescued the axon guidance defects of null orbit/MAST alleles. Thus, Orbit/MAST is required cell autonomously during neuronal differentiation for accurate axon guidance decisions (Lee, 2004).
The late-stage axon pathway defects in orbit/MAST mutants suggest a failure in the repellent effects of Slit on growth cone orientation. To be certain that the orbit/MAST phenotype reflects a loss of growth cone orientation and not simply a change in patterns of axon fasciculation, axon trajectories of pioneer neurons was inspected before other axons were available to serve as a substrate for fasciculation. At late stage 12, the posterior corner cell (pCC) helps to pioneer the MP1 pathway proximal to the midline; pCC neurites extend anteriorly and slightly away from the midline in wild-type. In orbit/MAST homozygotes, the pCC often orients toward the midline, sometimes crossing to meet its contralateral homolog. This shows that Orbit/MAST is required for accurate directional specificity of axon growth (Lee, 2004).
In addition to controlling midline crossing of axons, Slit repulsion determines the lateral position of longitudinal axon fascicles within the CNS neuropil. A marker for a mediolateral axon fascicle (Sema2b-Tau-myc) was used to examine this later function of Slit and its Robo receptors. In wild-type, Sema2b-positive axons cross the midline, turn, and extend along a straight longitudinal trajectory. In orbit/MAST mutants, a few Sema2b-positive axons meandered toward the midline from lateral positions. However, measurement of the lateral separation of these axon tracts reveals a significant inward shift in orbit/MAST mutants. Together, these genetic data demonstrate that Orbit/MAST performs a cell-autonomous postmitotic function during growth cone navigation (Lee, 2004).
The interaction between Orbit/MAST and Abl in the retina predicted that these proteins might cooperate to mediate axon guidance choices. However, since Abl plays both positive and negative roles in Slit signaling, it was important to test the polarity of genetic interactions in the context of embryonic development. Abl and Orbit/MAST levels were elevated, alone or in combination in postmitotic neurons. A mild synergy between the two genes during midline guidance was found that is consistent with cooperation. Interestingly, overexpression of Orbit/MAST alone induces a low but significant number of guidance errors at the midline. Stronger interactions were observed through LOF analysis. Double homozygous LOF mutants showed substantially increased ectopic midline crossing compared to single mutant controls, reminiscent of mutations in robo itself. Due to large maternal contributions of Abl and Orbit/MAST, even amorphic alleles are not zygotic null. Thus, it is not possible to use the double LOF mutant to conclude that both proteins act in a common pathway; however, the observed synergy does show that Abl and Orbit/MAST cooperate during midline axon guidance (Lee, 2004).
Since Abl is also required for motor axon pathfinding in the periphery (Wills, 1999a), intersegmental nerve b (ISNb) morphology was compared in double and single mutants. Overexpression of Abl generates an ISNb bypass phenotype where this group of axons fail to enter their target domain (Wills, 1999b). Coexpression of Abl and Orbit/MAST does enhance the expressivity of phenotype slightly, but the effect is subtle. Once having entered the ventral target domain, wild-type ISNb axons innervate the clefts between muscles 6, 7, 12, and 13. In Abl LOF mutants, ISNb stops short of its final targets, often terminating at muscle 13 (Wills, 1999a). A similar ISNb growth cone arrest phenotype is observed at very low penetrance in orbit/MAST LOF alleles. However, comparison of these phenotypes to orbit,Abl recombinant homozygotes revealed a strong enhancement of ISNb arrest in double LOF mutants, increasing the frequency of defects and shifting arrest to a more proximal position at the muscle 6/7 cleft. Thus, Abl and Orbit/MAST cooperate during axon guidance decisions in multiple contexts (Lee, 2004).
Analysis of CNS axons suggested that Orbit/MAST is an effector in the Slit/Robo repellent pathway. To test the hypothesis, the same genetic assay was used that was used to identify Slit as the ligand for the Robo receptor family. While heterozygotes lacking one copy of Slit or its receptors show very few guidance errors at the midline choice point, transheterozygotes that also remove one copy of a second gene in the pathway often reveal strong, synergistic phenotypes. Indeed, while orbit/MAST heterozygotes show no significant midline defects, very strong synergy is observed with mutations in slit (roughly 10-fold). As a control for the specificity of the interaction, embryos were examined lacking different alleles of orbit/MAST and an allele of capulet (capt), an actin binding protein that shows strong interactions with both slit and Abl (Wills, 2002). No synergy was observed between capt and orbit/MAST. The same transheterozygote analysis was performed with single mutations in the repellent receptors; orbit/MAST was found to enhance robo. Additional crosses revealed that orbit/MAST interacts with robo and robo2 but not with robo3, consistent with the specialization of Robo and Robo2 for midline crossing. To be certain that Orbit/MAST is not required simply for the expression or delivery of Slit and/or Robo protein, staining in wild-type and orbit/MAST embryos was compared, but no obvious differences were seen (Lee, 2004).
While all the data supported the model that Orbit/MAST is necessary for Abl function during axon guidance, a more rigorous test was desired. If Orbit/MAST acts as an effector of Abl, orbit/MAST mutations would be expected to be epistatic to an Abl GOF phenotype. The fact that Abl acts in both positive and negative capacities during midline guidance complicates the interpretation of such an experiment within the CNS; however, Abl plays a less complex role for ISNb motor axons (Wills, 1999b). When overexpressed under a strong postmitotic neural GAL4 source, Abl generates an ISNb bypass phenotype; neuronal expression of GAL4 alone has no effect. However, when Abl is overexpressed in an orbit/MAST homozygous background, the frequency of ISNb bypass drops approximately 2-fold. This indicates that Orbit/MAST acts genetically downstream of Abl in embryonic growth cones (Lee, 2004).
The relative roles of astral and central spindle microtubules (MTs) in cytokinesis of Drosophila melanogaster primary spermatocytes have been assessed. Time-lapse imaging studies reveal that the central spindle is comprised of two MT populations: 'interior' central spindle MTs found within the spindle envelope and 'peripheral' astral MTs that probe the cytoplasm and initiate cleavage furrows where they contact the cortex and form overlapping bundles. The MT-associated protein Orbit/Mast/CLASP concentrates on interior rather than peripheral central spindle MTs. Interior MTs are preferentially affected in hypomorphic orbit mutants, and consequently the interior central spindle fails to form or is unstable. In contrast, peripheral MTs still probe the cortex and form regions of overlap that recruit the Pav-KLP motor and Aurora B kinase. orbit mutants have disorganized or incomplete anillin and actin rings, and although cleavage furrows initiate, they ultimately regress. This work identifies a new function for Orbit/Mast/CLASP and identifies a novel MT population involved in cleavage furrow initiation (Inoue, 2004).
The terminal event of cell division, cytokinesis, is driven by the constriction of an actomyosin 'contractile' ring. However, the mechanisms responsible for determining the cleavage site remain poorly understood. Data from a variety of systems have implicated the spindle, a bipolar microtubule (MT) structure, in dictating furrow position. Three models have been put forward to explain how the spindle and its component MTs might signal furrow formation. The first is exemplified from experiments in which cells containing multiple spindles enter cytokinesis. In systems as diverse as echinoderm eggs and cultured vertebrate somatic cells, cleavage furrows initiate at the spindle equators as expected, as well as between the adjacent spindles. These observations raised the possibility that astral MTs provide a positive signal for furrow positioning and induction. In contrast, a second model suggests a negative regulatory role for astral MTs. Here, the polar regions of a cell would be less contractile due to the higher density of astral MTs. In agreement with this finding, it has been noted that in C. elegans embryos, furrows initiate at locations where the MT density is lowest. A third model, that accommodates physical and genetic data, attributes furrow positioning to the central spindle, a dense array of overlapping MTs that forms during late anaphase. For example, placement of a barrier between one side of the central spindle and the cell cortex results in furrow formation only on the nonobstructed side. Gatti (2000) showed that the central spindle is necessary and sufficient for furrow formation in Drosophila cells. Mutations that greatly diminish astral MTs without apparently affecting the central spindle do not inhibit cytokinesis as revealed by fixed meiotic and mitotic cells. Furthermore, loss of central spindle integrity by mutation or RNAi leads to polyploid cells (Inoue, 2004 and references therein).
Several studies in C. elegans embryos have shown that the central spindle is dispensable for furrow initiation but is involved in its propagation for midbody formation. Interestingly, when anaphase B spindle elongation is genetically restricted in these cells, a dependence on the central spindle for furrow initiation is observed. Thus, the roles that the different MTs serve in cytokinesis remains unclear (Inoue, 2004).
This problem is being approached by examining the effects of mutations on a variety of MT-associated proteins (MAPs) during the onset of cytokinesis in Drosophila primary spermatocytes. This study examines the role of the Orbit/Mast protein. Drosophila orbit/mast mutants were first identified through genetic screens as displaying a variety of mitotic defects including the formation of both mono- and multipolar spindles. Time-lapse observations of mast mutant embryos supported by RNAi studies on cultured cells showed that the gene product is needed to maintain spindle bipolarity and chromosome congression (Maiato, 2002). The Orbit/Mast protein has its counterpart in mammalian cells in the form of the CLASPs (Akhmanova, 2001), two related proteins identified through their ability to interact with CLIP-170/CLIP-115 proteins that associate with MT plus ends (Inoue, 2004 and references therein).
A recent study of the mitotic role of CLASP1 using antibody microinjection into cultured mammalian cells revealed a similar phenotype as that seen following RNAi of Orbit/Mast in Drosophila S2 cells (Maiato, 2002 & 2003). Monopolar spindles formed in which the chromosomes became buried in the interior of the monoaster. In both cases, treatment with MT stabilizing drugs caused the chromosomes to move to the plus ends of the MTs at the astral periphery. This finding was interpreted to indicate a role in regulating MT dynamics, specifically the transition from shrinkage to growth of the kinetochore MTs that link each chromosome to the spindle. The localization of both Orbit/Mast and CLASPs to the kinetochore is consistent with the above postulated role. Orbit/Mast also associates with the central region of late mitotic spindles in both Drosophila and mammalian cells (Inoue, 2000; Lemos, 2000; Maiato, 2003), as well as the ring canals and fusome of oocytes (Máthé, 2003). However, the metaphase arrest associated with the aforementioned spindle and chromosome congression defects has prevented the roles of Orbit/Mast during cytokinesis from being determined (Inoue, 2004).
This study provides the first characterization of central spindle formation in living Drosophila primary spermatocytes. The central spindle consists of two distinct sets of MTs, 'peripheral' astral MTs and 'interior' MTs that are confined within the spindle envelope, the highly fenestrated remains of the nuclear membrane that persist in Drosophila cells. These MT populations appear biochemically distinct since they show a differential association with the MAP encoded by orbit/mast. Time-lapse imaging reveals that the future cleavage site corresponds to locations where peripheral MTs contact the cortex and then bundle together. Furrows initiate and ingress, thereby coalescing peripheral and interior MTs to consolidate the late central spindle. The weak metaphase spindle integrity checkpoint in Drosophila primary spermatocytes provides the opportunity to examine late division events in mutants that would normally undergo metaphase arrest in the mitotic cells of somatic tissues (e.g., Polo kinase and Asp). In hypomorphic orbit mutants, peripheral MTs still contact the cortex and bundle, but interior MTs often fail to organize or are unstable and cleavage fails. In agreement with this phenotype, it is shown that in wild-type cells the Orbit/Mast protein concentrates on the spindle, but after anaphase onset selectively accumulates in the region described as the spindle matrix, which is occupied by interior but not peripheral MT bundles (Inoue, 2004).
The ongoing debate about the relative contribution of astral and central spindle MTs in the positioning of the cleavage furrow led to the examination of the dynamics of MT behavior in living Drosophila primary spermatocytes. To this end, a transgenic line ubiquitously expressing EGFP-tagged ß-tubulin was used. These otherwise wild-type cells were followed by multidimensional, near simultaneous, differential interference contrast (DIC) and fluorescence, time-lapse microscopy. Before anaphase onset, the polar regions of the cytoplasm contained numerous astral MTs, some of which appeared to be separated from the centrosomes. The post-anaphase spindle was found to contain two distinct populations of MTs, a peripheral set of long astral MTs originating from the polar regions of the cell, which became more robust and dynamic as they 'probed' the cytoplasm reaching toward the equator, and an interior set found within the remnants of the nuclear envelope, which appeared to elongate from the spindle poles, but did not extend into the equatorial region. Shortly thereafter, the peripheral MTs formed protrusions that contacted the cortex and formed bundles. The interior and most of the peripheral MTs then appeared to be released and translocated to the spindle equator, leaving behind a denuded area at the polar regions. Concomitantly the peripheral and interior MTs formed independent bundles at the equator appearing as a broad central spindle. Cleavage initiated 9 ± 1 min after anaphase onset, after the peripheral MTs from the two poles had contacted the cell cortex. These peripheral- and interior-central spindle MT bundles ultimately compact into a common midbody as the cleavage furrow ingresses (Inoue, 2004).
Therefore, this study of MT behavior during the early stages of cytokinesis in living Drosophila primary spermatocytes has identified two classes of central spindle MTs. The first corresponds to the peripheral MTs of the asters, which dynamically probe the cytoplasm as they extend along the cell periphery. This probing continues until the MTs contact the cortex and bundle at the location of the future cleavage site. These MTs translocate toward the spindle equator and form a series of overlapping bundles, a structure that has been termed the 'peripheral central spindle.' These are distinct from the interior MTs, which are located within the spindle envelope and appear to elongate and then appear to be released from the spindle poles to form the overlapping bundles of the 'interior central spindle.' It has been known for some time that cytokinesis in Drosophila spermatocytes requires the integrity of the central spindle MTs. However, the present study assigns the peripheral central spindle MTs the role of initiating furrow formation where they contact the equatorial cortex to form bundles, and a second function to the interior central spindle MTs to stabilize and propagate the furrow. In agreement with these findings, recent observations of Drosophila primary spermatocytes mutant for the abnormal spindle (asp) MT binding protein also implicate astral MTs in furrow placement. In these cells, the asters position themselves independently of an acentrosomally derived spindle. During cytokinesis, furrows initiate at locations between the two asters irrespective of the spindle axis. Along with this functional difference, the data suggest that peripheral and interior MTs are biochemically distinct, since the Orbit/Mast protein strongly accumulates in the region of the putative spindle matrix in the vicinity of the interior MTs, whereas it is not detectable above background levels on the regions containing the peripheral MTs. Interior MTs are preferentially affected in orbit mutants and fail to form stable interior central spindles; subsequently, cleavage furrows may initiate but not be sustained, leading to cytokinetic failure (Inoue, 2004).
The bundling of peripheral MTs simultaneous with or shortly after they contact the cortex but before furrowing onset raises the possibility that either of these events could be essential for furrowing. Successful cytokinesis has been shown to correlate with the presence of a central spindle-like structure even in the absence of canonical bipolar spindles. For example, when two spindles share a common cytoplasm in PtK1 cells at anaphase, not only does each spindle develop a mid-zone comprised of overlapping MTs and associated proteins, but similar structures form between the asters of the adjacent spindles. Moreover, central spindle-like structures can form from the monopolar spindles that result when either gamma-tubulin or an associated protein is mutated in Drosophila primary spermatocytes. These pseudo-central spindles contain the characteristic cleavage proteins and are functional, leading to asymmetrical cleavage with polyploid and anucleate daughter cells (Inoue, 2004).
Live cell analyses of S2 cells expressing GFP-tubulin suggest that contact of peripheral astral MTs with the cortex, rather than their bundling, is required for cleavage furrow positioning and onset. In these cells, long astral MTs emanating from either a single or the two opposing centrosomes contact the cleavage site near the time of furrow initiation. Similarly, it has been found that in cultured mammalian cells induced to form monopolar spindles by treatment with monastral, a subpopulation of stable MTs extends past the chromosomes and contacts the cortex at the site of furrow formation. Together these data suggest that it is the presence of astral MT plus ends and not their overlapping configuration that is critical for furrow placement (Inoue, 2004).
In apparent contradiction to these observations, cleavage can occur in the absence of astral MTs. Drosophila asterless mutants form central spindles and cleave, and yet asters were not detected in fixed preparations. Moreover, when the asters were removed from living grasshopper spermatocytes during anaphase by microsurgery, both the now asterless main cell and aster-containing cell derivative undergo cytokinesis. A fixed cell study of MT and actin distribution in these 'asterless' cells during furrow ingression revealed the presence of cortically associated MT bundles. It would be of interest to directly study how MTs form the central spindle under such asterless conditions in living cells (Inoue, 2004).
These data could be reconciled if both the peripheral/astral and interior MTs were able to deliver the same positive regulatory signal but that the peripheral MTs were more efficient at doing so. Thus, in normal wild-type cells, initially dynamic peripheral/astral MTs would contact the cortex and induce furrowing, while subsequent interactions between the interior MTs and the nascent contractile ring would then stabilize the furrow to allow for its propagation and midbody formation (Inoue, 2004).
A likely candidate for such a positive regulator is 'centralspindlin'. This highly conserved complex is comprised of an MKLP1 family member associated with a Rho-family GAP (MgcRacGAP in mammalian cells, CYK-4 in C. elegans, and RacGAP50C in Drosophila . The NH2-terminal region of RacGAP50C has also been recently shown to associate with the NH2 terminus of a conserved RhoGEF that in Drosophila is known as Pebble, a protein required to initiate furrow formation. Pebble activates RhoA and may promote actin polymerization and activation of Rho-dependent and Citron kinases. However, this model has been questioned in systems containing dense asters like C. elegans embryos and also in mammalian cells where overexpression of the NH2-terminal domain of the Pebble orthologue, ECT2, causes late defects in cytokinesis. Resolution of this issue awaits an understanding of the dynamics of furrow propagation, a process likely to be complex and involve cycles of activation and inactivation of the contractile machinery. In this light it will be of future interest to determine the relative parts played by the Pebble/ECT2 Rho GEF and the Rho-family GAP (Inoue, 2004).
Although the peripheral and interior central spindle MTs share several common features particularly with respect to their association with the centralspindlin complex, the present study reveals some differences. These data suggest Orbit/Mast may be required to promote growth and stability specifically of the interior central spindle, which forms where the protein preferentially associates. This adds another function to those already shown for this MAP that in early M-phase is needed to maintain spindle bipolarity and to facilitate chromosome congression along MT plus ends (Inoue, 2000; Lemos, 2000; Maiato, 2002). Those authors proposed that the protein promotes kinetochore fiber stability by facilitating the transition between MT shrinkage and growth at the kinetochore. Orbit/Mast may serve a similar role in stabilizing interior central spindle MTs. The diminution of such a MT-stabilizing protein is consistent with the rapid degradation of the interior MTs. It is unclear if the failure to release MTs from the spindle poles in orbit mutants is a direct effect or the result of less stable MT plus ends. It is speculated that peripheral MTs may require different dynamic properties than those of the interior to allow them to probe the exceptionally large volume of cytoplasm associated with spermatocytes and establish a furrow initiation site. This speculation raises the possibility that the peripheral MTs might use alternative MAPs to establish their zone of overlap (Inoue, 2004).
The work sheds new light on the question of whether or not cytokinesis is directed by astral or central spindle MTs. The observation that peripheral MTs specifically invade the equatorial cortex and become bundled at the site of furrow initiation conflicts with the hypothesis that cytokinesis occurs at regions of low MT density. It is easier to accommodate the observations into a model in which MTs supply a positive regulator of cytokinesis (or a negative regulator of some other inhibitor) rather than providing a means of concentrating inhibitory molecules (Inoue, 2004).
The motion of a chromosome during mitosis is mediated by a bundle of microtubules, termed a kinetochore fibre (K-fibre), which connects the kinetochore of the chromosome to a spindle pole. Once formed, mature K-fibres maintain a steady state length because the continuous addition of microtubule subunits onto microtubule plus ends at the kinetochore is balanced by their removal at their minus ends within the pole. This condition is known as 'microtubule poleward flux'. Chromosome motion and changes in position are then driven by changes in K-fibre length, which in turn are controlled by changes in the rates at which microtubule subunits are added at the kinetochore and/or removed from the pole. A key to understanding the role of flux in mitosis is to identify the molecular factors that drive it. Drosophila S2 cells expressing alpha-tubulin tagged with green fluorescent protein, RNA interference, laser microsurgery and photobleaching were used to show that the kinetochore protein MAST/Orbit - the single CLASP orthologue in Drosophila - is an essential component for microtubule subunit incorporation into fluxing K-fibres (Maiato, 2005).
The metaphase-spindle steady-state length occurs as spindle microtubules 'flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. This study used RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. It is concluded that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance (Laycock, 2006).
Bipolar spindle formation and maintenance occur through the actions of multiple motor and microtubule dynamics-altering proteins. This study examined the interplay between Orbit, a protein needed for tubulin-dimer incorporation into kinetochore MTs, and each of the four microtubule depolymerising kinesins Klp67A, Klp10A, Klp59C and Klp59D. The co-depletion of Klp10A but not of the other KLP MT depolymerases diminished the number of apoptotic cells and prevented the spindle collapse associated with orbit knockdown. In contrast to individual downregulation of Orbit or Klp10A that resulted in abnormally short and/or monopolar or long spindles, respectively, spindles in double-deficient cells were bipolar and of an average length indistinguishable from controls. These spindles promoted chromosome alignment and anaphase entry, indicating that they were functional. Since both Orbit and Klp10A have been directly implicated in microtubule flux, these data suggest that this process is not required to determine mitotic spindle morphology or chromosome congression in Drosophila tissue culture cells (Laycock, 2006).
During the course of this work, it was reported that microtubule flux is not essential for bipolar spindle formation and chromosome congression in mitotic vertebrate cells. In contrast to the knocked down MT-stabilising and -depolymerising protein pair described in this study, the experiments in vertebrate used the co-depletion of two kinesin-13 depolymerases: Kif2A, the orthologue of Klp10A and Kif2C/MCAK/XKCM1, the vertebrate counterpart of Klp59C. Both Kif2A and Kif2C have overlapping localisations at centromeres and spindle poles and/or centrosomes and loss of either leads to a prometaphase spindle collapse. This monopolar spindle phenotype was rescued by simultaneously depleting both of these MT depolymerases, suggesting they form an antagonistic pair. However, an orthologous antagonism of Klp10A and Klp59C does not exist during mitosis in Drosophila, because loss of Klp59C function in tissue culture cells or syncitial embryos does not alter spindle morphology, and dual perturbations result in spindle abnormalities identical to that observed following single Klp10A disruptions (Laycock, 2006 ans references therein).
It is proposed that bipolar spindle formation occurs in at least two phases. During the first, MTs nucleated from the separated centrosomes invade the nuclear volume and make their initial interactions with the kinetochores. Time-lapse analyses indicate that this step is not affected by the depletion of either Orbit or Klp10A and is thus Orbit- and Klp10A-independent. The second phase, spindle stabilisation, occurs after chromosome bi-orientation and probably results from antagonistic pairs of molecules regulating the dynamics of the plus- and minus-ends of kinetochore MTs. It is at this stage that Orbit and Klp10A become engaged as evidenced by the collapse of the nascent spindle following their individual perturbations. Although the possibility that the spindle collapse results from a loss of interpolar MT integrity cannot be ruled out, this is thought to be unlikely. First, because the rate at which spindles collapse after orbit RNAi is similar to the rate of MT flux in Drosophila tissue culture cells, consistent with a flux depolymerase shortening kMTs in the absence of new tubulin-polymer growth at the kinetochore. Second, because kMTs that form independently from the centrosome and primary spindle axis (i.e. a mini-spindle) also shrink in the absence of Orbit. Since these bundles of kMTs are not present between two half spindles it unlikely that their shortening is the result of intervening interpolar MTs. Likewise, it has been previously demonstrated that the spindle collapse associated with the loss of Kif2A is kMT-dependent. Spindle bipolarity was restored in these cells by co-depleting the Nuf2 kinetochore protein, thereby preventing kMT formation without affecting any other spindle MTs. In the case of Orbit and Klp10A single-depleted cells, it is envisaged that the spindle collapse is due to an imbalance of regulatory components following the activation of the flux machinery during this second phase. Collapse could result, for example, as Klp10A depolymerises the non-polymerising kMTS that result from orbit downregulation. Conversely, because flux-generated tension has been proposed to promote tubulin subunit incorporate at kinetochores, depletion of Klp10A could also affect polymerisation of kMTs, which - in the presence of other active phase two depolymerises - would cause spindle collapse. In the absence of Orbit and Klp10A the flux machinery would not become engaged and spindle length would be determined by other antagonistic molecular pairs (Laycock, 2006).
The data in flies, in concert with that found in vertebrates, indicate that although microtubule flux is a characteristic of many animal cells it is not essential for pre-anaphase chromosome movements or spindle formation. Nevertheless, the plasticity it imparts is probably advantageous for spindle and kinetochore interactions, for example by promoting kMT polymerisation or by generating tension for satisfying the spindle checkpoint. It was found that cells simultaneously depleted of Klp10A and Orbit tended to spend variable but extremely prolonged periods of time in mitosis before entering anaphase. Since both fixed and live cell studies did not reveal an increase in non-equatorially positioned chromosomes compared with controls, it is believed that the prometaphase arrest was not due to activation of the checkpoint through unattached kinetochores. Although never fully relaxed, the centromeres of bi-oriented chromosomes in double-knockdown cells tended to be under diminished tension relative to controls. This corresponded to the retention of BubR1 at kinetochores. Cells depleted only of Klp10A also spent more time in prometaphase than their control counterparts, although this duration was substantially less than that observed for orbit and Klp10A double-RNAi cells. Despite decreased intra-centromeric tension in Klp10A downregulated cells, BubR1 was not observed on the kinetochores of congressed chromosomes. One explanation for this is that, even without flux, spindle MTs can still produce tension by transducing cortical forces. Here, the long spindles that form in the absence of Klp10A would position the asters in direct contact with the cortex where their component MTs would make increased numbers of contacts with cortical motor proteins such as cytoplasmic dynein. Just as cortex-based forces by this motor act along astral MTs for spindle positioning, astral pulling could generate tension across the centromeres and kinetochores of congressed chromosomes. If true, centromeric tension should correlate with the presence or absence of asters. This was found to be the case, and sister centromeres were separated to a greater extent when they were on bi-astral spindles than when attached to bipolar spindles with a single aster. Moreover, in those Klp10A-depleted cells displaying bipolar spindles capped at each end by an overgrown aster, the average intra-centromeric distance was greater than that seen in the controls (Laycock, 2006).
Together, these observations indicate that Orbit and Klp10A are an antagonistic molecular pair, consistent with their previous individual implicated roles in MT flux, a process that dispensable for bipolar spindle formation and chromosome congression. These data further suggest that astral-mediated pulling forces are involved in checkpoint satisfaction. The role that these forces may serve in the checkpoint has not been previously reported (Laycock, 2006).
An important group of microtubule associated proteins are the plus-end tracking proteins which includes the Mast/Orbit/CLASPs family among others. Several of these proteins have important functions during interphase and mitosis in the modulation of the dynamic properties of microtubules, however, the precise mechanism remains to be elucidated. To investigate the role of Mast in the regulation of microtubule behaviour during interphase, RNAi was used in Drosophila S2 culture cells stably expressing GFP-α-tubulin and the behaviour of microtubules was followed in vivo. Mast depleted cells show a significant reduction of microtubule density and an abnormal interphase microtubule array that rarely reaches the cell cortex. Analysis of the dynamic parameters revealed that in the absence of Mast, microtubules are highly dynamic, constantly growing or shrinking. These alterations are characterized by a severe reduction in the transition frequencies to and from the pause state. Moreover, analysis of de novo microtubule polymerization after cold treatment showed that Mast is not required for nucleation since Mast depleted cells nucleate microtubules soon after return to normal temperature. Taken together these results suggest that Mast plays an essential role in reducing the dynamic behaviour of microtubules by specifically promoting the pause state (Sousa, 2007).
Akhmanova, A., Hoogenraad, C. C., Drabek, K., Stepanova, T., Dortland, B., Verkerk, T., Vermeulen, W., Burgering, B. M., De Zeeuw, C. I., Grosveld, F. and Galjart, N. (2001). Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts. Cell 104: 923-935. 11290329
Bashaw, G. J., Kidd, T., Murray, D., Pawson, T. and Goodman, C. S. (2000). Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor. Cell 101: 703-715. 10892742
Brittle, A. L., and Ohkura, H. (2005). Mini spindles, the XMAP215 homologue, suppresses pausing of interphase microtubules in Drosophila. EMBO J. 24: 1387-1396. PubMed Citation: 15775959
Brouhard, G. J., et al. (2008). XMAP215 is a processive microtubule polymerase. Cell 132: 79-88. PubMed Citation: 18191222
Brunner, D., and P. Nurse. (2000). CLIP170-like tip1p spatially organizes microtubular dynamics in fission yeast. Cell 102: 695-704. 11007487
Carvalho, P., Tirnauer, J. S. and Pellman, D. (2003). Surfing on microtubule ends. Trends Cell Biol. 13: 229-237. 12742166
Engel, U., Zhan, Y., Long, J. B., Boyle, S. N., Ballif, B. A., Dorey, K., Gygi, S. P., Koleske, A. J. and Van Vactor, D. (2014). Abelson phosphorylation of CLASP2 modulates its association with microtubules and actin. Cytoskeleton (Hoboken) [Epub ahead of print]. PubMed ID: 24520051
Fedorova, S. A., Chubykin, V. L., Gusachenko, A. M. and Omel'yanchuk, L. V. (1997). Mutation chromosome bows (chbv40), associated with the abnormal chromosome spindle in Drosophila melanogaster. Genetika 33: 1502-1509 (published in English in Russ. J. Genet. 33(11): 1286-1292). 9480214
Gatti, M., Giansanti, M. G. and Bonaccorsi, S. (2000). Relationships between the central spindle and the contractile ring during cytokinesis in animal cells. Microsc. Res. Tech. 49: 202-208. 10816260
Goshima, G., Wollman, R., Stuurman, N., Scholey, J. M. and Vale, R. D. (2005). Length control of the metaphase spindle. Curr. Biol. 15(22): 1979-88. Medline abstract: 16303556
Grevengoed, E. E., Loureiro, J. J., Jesse, T. L. and Peifer, M. (2001). Abelson kinase regulates epithelial morphogenesis in Drosophila. J. Cell Biol. 155: 1185-1198. 11756472
Hsouna, A., Kim, Y. S. and VanBerkum, M.F. (2003). Abelson tyrosine kinase is required to transduce midline repulsive cues. J. Neurobiol. 57: 15-30. 12973825
Inoue, Y. H., do Carmo Avides, M., Shiraki, M., Deak, P., Yamaguchi, M., Nishimoto, Y., Matsukage, A. and Glover, D. M. (2000). Orbit, a novel microtubule-associated protein essential for mitosis in Drosophila melanogaster. J. Cell Biol. 149: 153-166. 10747094
Inoue, Y. H., Savoian, M. S., Suzuki, T., Mathe, E., Yamamoto, M. T. and Glover, D. M. (2004). Mutations in orbit/mast reveal that the central spindle is comprised of two microtubule populations, those that initiate cleavage and those that propagate furrow ingression. J. Cell Biol. 166(1): 49-60. 15240569
Jones, M. H., Bachant, J. B., Castillo, A. R. Giddings, T. H. and Winey M. (1999). Yeast Dam1p is required to maintain spindle integrity during mitosis and interacts with the Mps1p kinase. Mol. Biol. Cell. 10(7): 2377-91. 10397771
Komarova, Y. A., Akhmanova, A. S., Kojima, S., Galjart, N. and Borisy, G. G. (2002). Cytoplasmic linker proteins promote microtubule rescue in vivo. J. Cell Biol. 159: 589-599. 12446741
Laycock, J. E., Savoian, M. S. and Glover, D. M. (2006). Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo. J. Cell Sci. 119(Pt 11): 2354-61. 16723741
Lee, H. and Van Vactor, D., (2003). Neurons take shape. Curr. Biol. 13, pp. R152-R161. 12593820
Lee, H., et al. (2004). The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance. Neuron 42: 913-926. 15207236
Lemos, C. L., Sampaio, P., Maiato, H., Costa, M., Omel'yanchuk, L. V., Liberal, V. and Sunkel, C. E., (2000). Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization. EMBO J. 19: 3668-3682. 10899121
Lin, H., et al. (2001). Polyploids require Bik1 for kinetochore-microtubule attachment. J. Cell Biol. 155: 1173-1184. 11756471
Lowery, L. A., et al. (2010). Parallel genetic and proteomic screens identify Msps as a CLASP-Abl pathway interactor in Drosophila. Genetics 185(4): 1311-25. PubMed Citation: 20498300
Maiato, H., Sampaio, P., Lemos, C. L., Findlay, J., Carmena, M., Earnshaw, W. C. and Sunkel, C. E. (2002). MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity. J. Cell Biol. 157: 749-760. 12034769
Maiato, H., Fairley, E. A., Rieder, C. L., Swedlow, J. R., Sunkel, C. E. and Earnshaw, W. C. (2003a). Human CLASP1 is an outer kinetochore component that regulates spindle microtubule dynamics. Cell 113(7): 891-904. 12837247
Maiato, H., Rieder, C. L, Earnshaw, W. C. and Sunkel, C. E. (2003b) How do kinetochores CLASP dynamic microtubules? Cell Cycle 2(6): 511-4. 14504462
Maiato, H., Khodjakov, A. and Rieder, C. L. (2005). Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres. Nat. Cell Biol. 7: 42-47. 15592460
Máthé, E., Inoue, Y. H., Palframan, W., Brown, G. and Glover, D. M. (2003). Orbit/Mast, the CLASP orthologue of Drosophila, is required for asymmetric stem cell and cystocyte divisions and development of the polarised microtubule network that interconnects oocyte and nurse cells during oogenesis. Development 130(5): 901-15. 12538517
Miller, A. L., Wang, Y., Mooseker, M. S. and Koleske, A. J. (2004). The Abl-related gene (Arg) requires its F-actin-microtubule cross-linking activity to regulate lamellipodial dynamics during fibroblast adhesion. J. Cell Biol. 165: 407-419. 15138293
Moresco, E. M. and Koleske, A. J. (2003). Regulation of neuronal morphogenesis and synaptic function by Abl family kinases. Curr. Opin. Neurobiol. 13: 535-544. 14630215
Pasqualone, D., and Huffaker, T. C. (1994). STU1, a suppressor of a ß-tubulin mutation, encodes a novel and essential component of the yeast mitotic spindle. J. Cell Biol. 127: 1973-1984. 7806575
Persico, V., Callaini, G. and Riparbelli, M. G. (2019). The male stem cell niche of Drosophila melanogaster: Interactions between the germline stem cells and the hub. Exp Cell Res 383(1): 111489. PubMed ID: 31278899
Popov, A. V. and Karsenti, E. (2003). Stu2p and XMAP215: turncoat microtubule-associated proteins? Trends Cell Biol. 13: 547-550. PubMed Citation: 14573345
Shoda, T., Yamazoe, K., Tanaka, Y., Asano, Y. and Inoue, Y. H. (2021). Orbit/CLASP determines centriole length by antagonising Klp10A in Drosophila spermatocytes. J Cell Sci. PubMed ID: 33674447
Sousa, A., et al. (2007). The Drosophila CLASP homologue, Mast/Orbit regulates the dynamic behaviour of interphase microtubules by promoting the pause state. Cell Motil. Cytoskeleton 64: 605-620. PubMed Citation: 17487886
Stepanova, T., Slemmer, J., Hoogenraad, C.C., Lansbergen, G., Dortland, B., De Zeeuw, C.I., Grosveld, F., van Cappellen, G., Akhmanova, A. and Galjart, N. (2003). Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein). J. Neurosci. 23: 2655-2664. 12684451
Trogden, K. P. and Rogers, S. L. (2015). TOG proteins are spatially regulated by Rac-GSK3β to control interphase microtubule dynamics. PLoS One 10: e0138966. PubMed ID: 26406596
Tsvetkov, A. S., et al. (2007). Microtubule-binding proteins CLASP1 and CLASP2 interact with actin filaments. Cell Motil. Cytoskeleton 64: 519-530. PubMed Citation: 17342765
Watanabe, T., et al. (2009). Phosphorylation of CLASP2 by GSK-3beta regulates its interaction with IQGAP1, EB1 and microtubules. J. Cell Sci. 122: 2969-2979. PubMed Citation: 19638411
Wills, Z., Marr, L., Zinn, K., Goodman, C. S. and Van Vactor, D. (1999a). Profilin and the Abl tyrosine kinase are required for motor axon outgrowth in the Drosophila embryo. Neuron 22: 291-299. 10069335
Wills, Z., Bateman, J., Korey, C., Comer, A. and Van Vactor, D., (1999b). The tyrosine kinase Abl and its substrate Enabled collaborate with the receptor phosphatase Dlar to control motor axon guidance. Neuron 22: 301-312. 10069336
Wills, Z., Emerson, M., Rusch, J., Bikoff, J., Baum, B., Perrimon, N. and Van Vactor, D. (2002). A Drosophila homolog of cyclase-associated proteins collaborates with the Abl tyrosine kinase to control midline axon pathfinding. Neuron 36: 611-622. 12441051
Yin, H., You, L., Pasqualone, D., Kopski, K. M. and Huffaker, T. C. (2002). Stu1p is physically associated with beta-tubulin and is required for structural integrity of the mitotic spindle. Mol. Biol. Cell. 13(6): 1881-92. 12058056
date revised: 25 August 2021
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.